title: Synthetic substrates specific to activated plasmin can monitor the enzymatic functional status in situ in breast cancer cells.
abstract: We here strove to overcome the limitations of expression analyses such as PCR and IHC, based on molecular recognition between target and probe molecules, by designing synthetic substrates specific to the target molecules to directly estimate the enzymatic functionality in situ. The specific substrate contains a probing unit, which is an organic fragment for specific enzyme binding, and a reactive unit, which is a natural peptide subject to catalysis. In this study, the activation of plasminogen to plasmin was examined in MDA-MB231 breast cancer cells using the plasmin-specific synthetic substrates designed from their inhibitors. The localization and function of the activated plasmin were successfully visualized by fluorophore combined with the specific substrate concurrently. This would be the first time for activated plasmin at work in situ by direct observation. Our concept to directly monitor the functionality of target enzymes can be used straightforwardly for other proteases such as cathepsins or caspases. Also, this substrate concept as a 'tailor-made substrate' would be utilized as a novel functional molecular probe in vivo with appropriate detectable probes.
authors.affiliation: Computer-aided Molecular Modeling Research Center, Kansai (CAMM-Kansai), 5-1-7 Ohmichidori, Nagata-ku, Kobe, 653-0833, Japan.
authors.forename: Keigo
authors.initials: K
authors.lastname: Gohda
authors.forename: Ko
authors.initials: K
authors.lastname: Fujimori
authors.forename: Naoki
authors.initials: N
authors.lastname: Teno
authors.forename: Keiko
authors.initials: K
authors.lastname: Wanaka
authors.forename: Yuko
authors.initials: Y
authors.lastname: Tsuda
mesh_terms: D001665:Binding Sites; D001943:Breast Neoplasms / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D005260:Female; D005341:Fibrinolysin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D018613:Microscopy, Confocal; D062105:Molecular Docking Simulation; D010958:Plasminogen / Q000378:metabolism; D011480:Protease Inhibitors / Q000138:chemical synthesis* / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
chemical_list: D005456:Fluorescent Dyes; D011480:Protease Inhibitors; D010958:Plasminogen; D005341:Fibrinolysin
keywords: breast cancer; imaging probes; in situ functionality; plasminogen-plasmin system; specific synthetic substrates; structure-based molecular design